The invention pertains to systems for column-based separation, and to methods of forming and utilizing packed columns. In specific embodiments, the invention pertains to methods of separating sample components.
Column-based separations are frequently used for selectively removing components from mixtures. A first step in utilizing column-based technology is to form a column. Such can be accomplished within a column chamber. An exemplary prior art column chamber 10 is illustrated in FIG. 1. Column chamber 10 comprises a longitudinal tubular section 12 having ends 14 and 16. An inlet 18 is provided at end 16, and an outlet 20 is provided at end 14. Outlet 20 is obstructed by a porous filter 22. Filter 22 can comprise, for example, a porous fritted glass or ceramic material.
A packed column is formed within chamber 10 by flowing a slurry comprising a mixture of matrix material 15 and carrier fluid 17 into inlet 18. Matrix material 15 comprises a plurality of particulates, such as, for example, beads. Filter 22 is permeable to the carrier fluid and impermeable to the matrix material. Accordingly, as the slurry is flowed into column chamber 10, matrix material 15 stacks against filter 22 to form a packed column 19 within tubular portion 12.
The composition of carrier fluid 17 and matrix material 15 vary depending on the components that are intended to be separated by the packed column, and on the mixtures (samples) within which such components are found. Example matrix materials are Sr-resin, TRU-resin, and TEVA-resin, all of which can be obtained from EIChrom Industries, Inc., of Darien, Ill. Such matrix materials can have particle sizes in the range of, for example, 20-100 micrometers. Sr-resin, TRU-resin, and TEVA-resin can be used for, for example, selectively retaining radioactive materials. Specifically, Sr-resin can selectively retain strontium, TRU-resin can selectively retain americium, and TEVA-resin can selectively retain technetium. Slurries utilized for forming packed columns of Sr-resin, TEVA-resin, or TRU-resin can comprise, for example, 0.074 gram/mL of Sr-resin in 3 M HNO3; 0.142 grams/mL of TEVA-resin in 4 M HNO3; or 0.076 grams/mL of TRU-resin in 0.1 M HNO3, respectively.
Other materials that can be separated utilizing column-based systems are, for example, biological materials, such as nucleic acids. For instance, Tepnel Life Sciences sells polymeric micro-beads in diameters of approximately 60-100 micrometers which are covalently linked to specific oligonucleotide capture probes. Such micro-beads can be utilized for selective purification of nucleic acid fragments from a biological sample. For purposes of interpreting this disclosure and the claims that follow, the term xe2x80x9cnucleic acidxe2x80x9d is defined to include DNA nucleotides and RNA nucleotides, as well as any length polymer comprising DNA nucleotides or RNA nucleotides.
In addition to the above-discussed exemplary uses for column-based separations, numerous other applications for column-based separations are known to persons of ordinary skill in the art. The column-based separations generally have in common that a mixture in a first physical state (typically either a gas phase or a liquid phase) is flowed across a column matrix in a second physical state (typically either a liquid phase or a solid phase) to separate a component of the mixture from other materials of the mixture. Accordingly, the physical state of the matrix is generally different than the physical state of the component that is to be separated.
It can be desired to quantitate and/or otherwise analyze an amount of a component retained by a column matrix in a packed column. Accordingly, it can be desired to extract a retained component from a matrix materia. A method of extracting a retained component is to subject the column matrix to conditions which disrupt interactions between the matrix material and the component to thereby elute the component from the matrix material. In some applications, it is desirable to elute the retained material from the matrix material while the matrix material is still within a packed column, and in other applications it is desirable to remove the matrix material from, a packed column before eluting the retained component. Additionally, there are some applications in which it is desirable to remove a matrix material from a packed column and thereafter analyze the matrix material directly to quantitate and/or otherwise analyze an amount of a component retained on the matrix material.
A difficulty in utilizing column-based separations is in removing matrix material from a column chamber and subsequently repacking additional matrix material in the chamber to re-form a packed column. There are numerous reasons for removing matrix material from a chamber. For instance, a matrix material of a packed column can be rendered unusable after an initial separation, or after an initial series of separations. A matrix material can be rendered unusable if it is degraded by fluids passed through the material during a separation. Also, the matrix material can be rendered unusable if it becomes contaminated by materials within a sample because such contamination can pose a risk of cross-contamination.
For one or more of the above-discussed reasons, it is frequently desirable to repeatedly pack and unpack a column chamber with matrix material. Because packing and unpacking of column chambers is a time-consuming and laborious process, disposable columns are generally used. However, disposable columns still require labor for column changeout. Accordingly, it is desirable to develop new methods for packing and unpacking column chambers.
A recent improvement is described with reference to an apparatus 30 in FIGS. 2 and 3. Referring to FIG. 2, apparatus 30 comprises a tubular column chamber 32 having an inlet end 34 and an outlet end 36. Outlet end 36 terminates proximate a plate 38. Plate 38 can comprise a window configured to enable light to pass through for spectroscopic measurement of materials eluting from column chamber 30. A matrix material 40 forms a packed column 42 within column chamber 32. Packed column 42 has a lateral periphery defined by tubular chamber 32. Packed column 42 can be formed by flowing a slurry of matrix material 40 and a carrier fluid into column chamber 32. Outlet end 36 of column chamber 32 is displaced from plate 38 by a distance xe2x80x9cDxe2x80x9d sufficient to enable the carrier fluid to pass between column chamber 32 and plate 38. However, the distance is less than an average width of matrix material 40. Accordingly, matrix material 40 is retained in column chamber 32 and stacks against plate 38 to form packed column 42.
FIG. 3 illustrates a method for removal of matrix material 40 from packed column 42. Specifically, column chamber 32 is raised to enable matrix material 40 to pass beneath column chamber 32 and over plate 38. Subsequently, a fluid is flowed through chamber 32 to push matrix material 40 out of column chamber 32.
System 30 is improved relative to other methods of packing and unpacking columns in that it can provide a quick method for releasing packed column material from a column chamber, and can also provide a quick method for resetting the column chamber to be repacked with fresh matrix material. A difficulty with column system 30 is that it can be problematic to move an entirety of column chamber 32 during transitions between packing and unpacking operations. Further, precise tolerances are needed to hold beads and may leak beads. Discharged beads can undesirably pass through a detector. It can become increasingly difficult to move the entirety of column chamber 32 as a column-based separation is scaled up for larger operations. Accordingly, it is desirable to develop alternative methods for conveniently packing and unpacking column chambers, wherein a column chamber is not moved in transitioning between packing and unpacking operations.
In one aspect, the invention encompasses a method of packing and unpacking a column chamber. A matrix material is packed within a column chamber to form a packed column. After the packing, the matrix material is unpacked from the column chamber without moving the column chamber.
In another aspect, the invention encompasses a method of purifying a component of a sample. A column chamber having an inlet end and an outlet end is provided. The outlet end terminates proximate both a first flow, path and a second flow path. The first flow path is obstructed with a retaining material permeable to a first fluid and impermeable to a matrix material. The second flow path is blocked by a blocking material that removably blocks flow of both the first fluid and the column matrix material. A suspension of the first fluid and the matrix material is flowed into the column chamber and along the first flow path to form a packed column of the matrix material within the column chamber. The blocking material defines a portion of a periphery of the packed column. The matrix material is configured to selectively retain a component of the sample. The sample is flowed through the packed column and along the first flow path to separate the component from the rest of the sample. The blocking material is removed without moving the column chamber. After removing the blocking material, a second fluid is flowed through the column chamber and along .the second flow path to remove the matrix material from the column chamber.
In yet another aspect, the invention encompasses a system for column-based separations. The system comprises a fluid passageway containing a column chamber and a flow path in fluid communication with the column chamber. The flow path is obstructed by a retaining material permeable to a carrier fluid and impermeable to a column matrix material suspended in the carrier fluid. The flow path extends through the column chamber and through the retaining material. The flow path is configured to form a packed column within the column chamber when a suspension of the fluid and the column matrix material is flowed along the flow path. The column chamber defines a portion of a periphery configured to retain the packed column. Another portion of the periphery is defined by a blocking material that removably blocks flow of both the carrier fluid and the column matrix material. The blocking material is spaced from the packed column by a region configured to retain a fluid.
An advantage of the impermeable material is that the surface area of the material in contact with fluid is always in contact with fluid. In other words, there is no material surface area that alternately contacts fluid then, say an interior chamber surface. This feature minimizes the potential of sample to sample contamination since a sample may be completely washed through and not captured on an alternately or intermittently exposed surface. This is especially valuable for nucleic acid samples wherein one molecule of a previous nucleic acid sample can be detected in a subsequent nucleic acid sample.